Project Summary and Abstract Immune therapy for cancer has risen to prominence by successfully overcoming chemotherapy resistance. One of the most successful therapies is chimeric antigen receptor T cells (CAR) that target the CD19 protein on the surface of leukemia. Landmark clinical trials led by our group demonstrated unparalleled efficacy in relapsed and refractory acute lymphoblastic leukemia (ALL), with complete remission rates in excess of 80%, resulting in the first ever FDA approval of a gene modified cell therapy (Kymriah, tisagenlecleucel). There is a great need to develop CAR therapies for other pediatric cancers, and we face many challenges to achieve the same level of success in solid tumors and lymphoma. Primary among these challenges is the difficulty in collecting healthy T cells from pediatric cancer patients. Our experience not only treating the most children in the world with CAR therapy but also getting the most referrals gave us insight into the difficulty of getting healthy T cells from children with cancer. We have identified two main problems: dysfunction in T cells from children with solid tumors present at diagnosis and progressive difficulty getting functional T cells after intensive chemotherapy. As we have investigated these problems, we learned how fortunate we were with pediatric ALL, a patient group that has proven to have T cells uniquely well preserved for CAR therapy generation. This project seeks to identify the mechanisms of dysfunction in T cells from pediatric cancer patients both before and after chemotherapy, and reverse them to enable the generation of highly active CAR T cell therapies for cancers other than ALL. Our preliminary data indicates that Nave T cells are the key starting material for successful CAR T cell generation in pediatrics, and that in their absence preserving Nave-like function during CAR manufacture can substitute. Metabolic pathways are key in the transitions from Nave to memory and effector states, and these feed into the energy reserve of a T cell as measured by its mitochondrial potential. Our research plan centers on manipulating metabolic pathways and restoring mitochondrial respiratory capacity to T cells from pediatric cancer patients. This will enable the high level of proliferative capacity required for effective CAR therapy as we develop new targets within the Pediatric Immunotherapy Discovery and Development Network. Our work will support the discovery efforts of the PI-DDN by developing the specialized T cell manufacturing required for these difficult to treat cancers.